The present invention relates to the field of protecting groups in organic synthesis and, more particularly, to the use of these compounds as ribonucleoside protecting groups and as 2xe2x80x2-modifications. Still more specifically, the protecting groups are used in the synthesis of oligonucleotides containing ribonucleotide subunits.
The ability to routinely synthesize ribonucleic acid (RNA) has become increasingly important as research reveals the multitude of RNA""s biological functions. There are many types of RNA including ribosomal RNA, transfer RNA and messenger RNA. RNA also is important in various structures and functions as well as being a catalyst in enzymatic reactions, as in the case of ribozymes. Because of the important biological roles RNA plays, both known and unknown, it is of considerable utility to be able to synthesize short (2-300 nucleotides) defined sequences of RNA, commonly referred to as RNA oligonucleotides or oligoribonucleotides. Over the past 25 years, many chemical approaches have been explored for synthesizing RNA oligonucleotides. Because deoxyribonucleic acid (DNA) methodologies have progressed more rapidly, the usual strategy for the synthesis of RNA has been to adapt DNA chemistries to RNA synthesis. Consequently, most approaches have focused on retaining the 5xe2x80x2-dimethoxytrityl (DMT) ether and adding a compatible 2xe2x80x2-hydroxyl protecting group such as fluoride-labile silyl ethers, photo-labile moieties, and acid-labile acetals. A delicate balance has been required to successfully utilize the acid-labile 2xe2x80x2-acetals in conjunction with the acid-labile 5xe2x80x2-DMT ether. Therefore, other approaches have involved retaining the 2xe2x80x2-acetal while replacing the 5xe2x80x2-DMT.
The acid-labile acetals have many attractive features. For example, it has been reported that it is possible to chromatograph some 2xe2x80x2-acetal-protected RNA. However, the subsequent removal of these acetals, which are used with the DMT group, requires acidic conditions which subsequently cause degradation of the RNA, or require extremely long periods of time to remove ( greater than 24 hours). Therefore, although it may be possible to safely handle and purify some 2xe2x80x2-acetal-protected RNAs, the harsh conditions required for rapid deprotection may require further purification of the RNA, thereby negating this advantage. Milder conditions can be used but these are inconvenient, requiring more than 24 hours. More-labile acetals can not be used as they would not be sufficiently stable to the DMT acid-deprotection conditions during RNA synthesis.
Of all of the RNA synthesis methods reported to date, only the 5xe2x80x2-DMT-2xe2x80x2-t-butyldimethylsilyl (TBDMS) and the 5xe2x80x2-DMT-2xe2x80x2-[1-(2-fluorophenyl)-4-methoxypiperidin-4-yl] (FPMP) chemistries are readily available commercially. Unfortunately, neither of these methods allows RNA synthesis to be as routine and dependable as DNA synthesis. The impediments facing 5xe2x80x2-DMT-2xe2x80x2-FPMP chemistry are related to the problem of balancing two acid-labile protecting groups. One of the major difficulties with the 2xe2x80x2-TBDMS approach is that stepwise coupling yields are only 96-98% under routine conditions compared to  greater than 99% for DNA. These methods enable the synthesis of RNA in acceptable yields and quality, but a high level of skill and significant investments in training and experience are required to deliver adequate results.
One of the most desirable conditions for the final 2xe2x80x2-deprotection of synthesized RNA is an extremely-mildly-acidic aqueous solution. In the optimal scheme, the 2xe2x80x2-protecting groups only have to be stable to withstand oligonucleotide synthesis conditions. Scaringe and Caruthers (U.S. patent application Ser. No. 08/488,878, filed Jun. 9, 1995, now U.S. Pat. No. 5,889,136 and incorporated herein by reference) recently reported a novel RNA synthesis strategy similar to such a scheme. Their investigations led to the development of silyl ethers for protection of the 5xe2x80x2-hydroxyl. However, this 5xe2x80x2-silyl ether oligonucleotide synthesis chemistry was not compatible with mildly-acid-labile 2xe2x80x2-acetals. Acid-labile orthoester protecting groups were investigated and discovered to have potential for use at the 2xe2x80x2-hydroxyl. The 2xe2x80x2-orthoesters that were developed in conjunction with 5xe2x80x2-silyl ethers enabled the synthesis of RNA oligonucleotides. Scaringe and Caruthers disclose specific orthoester protecting groups at the 2xe2x80x2-position of ribonucleotides. However, they do not disclose the orthoesters of the present invention.
The present invention provides an orthoester moiety that serves as a protecting group, particularly for RNA synthesis. Also provided in this invention is RNA comprising the protecting group which possesses novel advantages and useful features, e.g., a modified RNA oligonucleotide that is easily handled and analyzed with minimal concern about degradation. The protecting groups can be readily cleaved ( less than 10 minutes), if so desired, under extremely mild conditions that cause no detectable degradation of the RNA. No prior art anticipates that the following 2xe2x80x2-modification 
would be advantageous to RNA synthesis. This 2xe2x80x2-modification is the result of using an orthoester of the following general structure: 
where R represents protecting groups which can be removed prior to removing the orthoester.
The prior art has provided several means to synthesize RNA oligonucleotides. However, none have enabled the synthesis, handling, analysis and use of RNA oligonucleotides to be as robust and dependable as DNA synthesis, nor produced RNA comparable to the high quality in which DNA can be produced. No prior art has disclosed a modification of RNA that enables the RNA to be easily handled and then where the modification, e.g., a protecting group, can be removed under mild conditions to yield fully deprotected RNA. The present invention, therefore, provides more robust RNA synthesis methods which consistently produce higher quality RNA on a routine basis.
Accordingly, it is an object of this invention to provide useful protecting groups for the improved synthesis, analysis, handling and use of RNA oligonucleotides or other polymers containing ribonucleotides. It is a further objective of this invention to provide RNA, comprising such protecting groups, that can be easily analyzed, handled and used without requiring extensive safeguards against degradation. The protecting groups can be subsequently removed if desired under extremely mild conditions to yield high-quality fully deprotected RNA. Those skilled in the art can use these protecting groups in other organic synthesis methodologies as well. Therefore, this invention is not limited to the field of nucleic acid and oligonucleotide chemistry.
The present invention achieves these and other objectives by the provision of novel orthoester protecting groups with innovative and useful features. We have developed, for example, the O-bis(2-acetyl-ethoxy)methyl (ACE) orthoester: 
that is stable to nucleoside and oligonucleotide synthesis conditions but is modified via ester hydrolysis during base deprotection of the oligonucleotide or polymer. The resulting 2xe2x80x2-bis(2-hydroxyethyl)methyl orthoester protecting group (2xe2x80x2-EG): 
is 10 times more labile to acid than it""s precursor, the ACE orthoester. Complete cleavage of the 2xe2x80x2-EG orthoester may be effected using extremely mild conditions (pH 3,  less than 10 min., 55xc2x0 C.). The innovative features of this chemistry have enabled the synthesis of RNA oligonucleotides surpassing results in the prior art in terms of quality.
The orthoesters of the present invention are illustrated as follows: 
where R represents protecting groups which can be removed prior to removing the orthoester. An example of an orthoester with appropriate R groups is as follows: 
where X1, X2, and X3 are appropriate atoms or ligands. Several suitable orthoesters are illustrated as follows: 
The general structure of a ribonucleoside of this invention is illustrated as follows: 
where exocyclic amines, 5xe2x80x2-hydroxyl and the 3xe2x80x2-hydroxyl groups are appropriately protected and/or functionalized for use in oligonucleotide synthesis and R represents protecting groups which can be removed while leaving the 2xe2x80x2-orthoester moiety intact. More specifically, the general structure of a ribonucleoside of this invention has the following structure where the R groups from above are acyl protecting groups and X1, X2, and X3 are appropriate atoms or ligands: 
The ACE orthoester comprises protecting groups. No prior art describes the use of protected orthoesters in oligonucleotide synthesis. It was not known that an orthoester utilizing, for example, protected ethylene glycol ligands would be stable to oligonucleotide synthesis conditions. It also was not known that orthoesters would be significantly more labile once protecting groups were removed from the ethylene glycol ligands. The novel orthoesters have been utilized for the synthesis of RNA of higher quality than that disclosed in prior art. Furthermore, this invention has made it possible to synthesize, handle, analyze and use RNA with ease comparable to DNA. Some useful aspects of this invention are as follows:
(1) It is possible to analyze and handle RNA while it is still 2xe2x80x2-protected. The structure of a typical ribonucleotide in a polymer of the present invention has the 2xe2x80x2-EG modified structure: 
The ability to analyze and handle polymers containing the 2xe2x80x2-EG-modified ribonucleotide subunit is important for two major reasons: (a) The 2xe2x80x2-modified RNA is relatively stable to degradation. Therefore, it is not necessary to observe stringent sterile conditions while handling, analyzing and purifying 2xe2x80x2-modified RNA synthesized using this invention. (b) While the RNA is still 2xe2x80x2-modified, it is possible to analyze and purify the oligonucleotides. Over 200 sequences have been synthesized according to this invention and it has been possible to resolve every oligonucleotide into a major product during analysis. (To those skilled in the art, it is known that a percentage of RNA oligonucleotides, approximately 5-10%, can not be resolved under routine analysis conditions if the RNA is fully deprotected because of strong secondary structures and folding common to RNA.) Thus, it is possible to synthesize RNA sequences without concern over whether it will be possible to analyze the final product.
(2) The RNA quality is consistent with any sequence For comparison, it has been reported that a 27-mer was synthesized with standard 5xe2x80x2-silyl-2xe2x80x2-orthoester chemistry in 35% overall yield. The same 27-mer was synthesized with 5xe2x80x2-DMT-2xe2x80x2-TBDMS chemistry in 45% overall yield. Using a novel orthoester of the invention, a comparable 27-mer was synthesized in  greater than 70% overall yield. A 36-mer described in a following example was routinely synthesized in 65-70% overall yield. Coupling yields of  greater than 99% were possible in  less than 90 seconds. For many applications using RNA synthesized with this invention, it is no longer necessary to purify the RNA after synthesis as the quality of the crude RNA synthesized is sufficient for subsequent use. The high quality and high yields of RNA observed with such short coupling times are comparable with those routinely experienced in DNA synthesis.
(3) The final 2xe2x80x2-deprotection conditions of the RNA oligonucleotide are the mildest ever reported for this application by an order of magnitude. For example, 2xe2x80x2-ACE-uridine has a half life of xcx9c7.5 minutes at pH 2, 25xc2x0 C., which is comparable to previously reported orthoesters and acetals that have been used in oligonucleotide synthesis. However, when the ACE orthoester is modified by ester hydrolysis, the 2xe2x80x2-modified uridine is 10 times more labile with a half life of  less than 45 seconds at pH 2, 25xc2x0 C. Several assays demonstrated that there was no detectable degradation or isomerization under the extremely mild conditions used to 2xe2x80x2-deprotect RNA oligonucleotides synthesized according to this invention.
This invention includes several other protecting groups. For example, the following 2xe2x80x2-acetal protecting group is included within this invention and can be synthesized by those skilled in the art utilizing the present specification and well-known synthetic techniques. 
This protecting group, or variations thereof, would exhibit similar properties to the orthoester protecting groups of this invention. Therefore, this invention includes other classes of protecting groups.
The present invention also provides polymers and oligonucleotides that comprise an acid-labile protecting group wherein the half life of the protecting group is as follows: the half life of that protecting group, when on the 2xe2x80x2-hydroxyl of a uridine nucleoside, is  less than 3 minutes at pH 2, 25xc2x0 C.
The invention also provides oligonucleotide comprising an acid-labile 2xe2x80x2-hydroxyl protecting group wherein the half life of the protecting group is as follows: the half life of that protecting group, when on the 2xe2x80x2-hydroxyl of a uridine nucleoside, is  less than 3 minutes at pH 2, 25xc2x0 C.
Further, the invention provides an oligonucleotide comprising an acid-labile 2xe2x80x2-hydroxyl protecting group which is itself protected by a second protecting group, and, wherein upon removal of the second protecting group(s) on the first protecting group, yield an acid-labile first protecting group wherein the half life of the first protecting group is now as follows: the half life of the deprotected first protecting group, when on the 2xe2x80x2-hydroxyl of a uridine nucleoside, is  less than 3 minutes at pH 2, 25xc2x0 C.
The features of the novel orthoesters of the present invention have made it possible to routinely synthesize RNA in high quality. Following synthesis, it is now possible to analyze almost any RNA oligonucleotide. Prior to this invention, this was not always possible. The RNA synthesized with this invention can be handled without the need for sterile conditions. When ready for use, the RNA is easily deprotected under extremely mild conditions that do not degrade the RNA nor contribute any detectable impurities. Oligonucleotides and polymers synthesized with this invention may be used for a wide array of purposes in various applications, including as antisense molecules, enzymatic molecules, diagnostic molecules, therapeutic molecules and research molecules. All of these uses are well known to those skilled in the art. The 2xe2x80x2-modification can be left on for subsequent applications, for example, analysis and purification, or it may be removed, if desired, to yield RNA with 2xe2x80x2-hydroxyl groups.